1. Field of the Invention
The present invention relates generally to fluorescent proteins, and more specifically to tandem fluorescent protein homodimers, which have a reduced propensity to oligomerize as compared to unlinked fluorescent protein monomers, and to methods of making and using such non-oligomerizing tandem fluorescent proteins.
2. Background Information
The identification and isolation of fluorescent proteins in various organisms, including marine organisms, has provided a valuable tool to molecular biology. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria, for example, has become a commonly used reporter molecule for examining various cellular process, including the regulation of gene expression, the localization and interactions of cellular proteins, the pH of intracellular compartments, and the activities of enzymes.
The usefulness of Aequorea GFP has led to the identification of numerous other fluorescent proteins in an effort to obtain proteins having different useful fluorescence characteristics. In addition, spectral variants of Aequorea GFP have been engineered, thus providing proteins that are excited or fluoresce at different wavelengths, for different periods of time, and under different conditions. The identification and cloning of a red fluorescent protein, dsRed, from Discosoma raised a great deal of interest due to its ability to fluoresce in the red wavelength. The availability of such fluorescent proteins has greatly expanded the studies that the proteins can be used for and, consequently, our understanding of cellular structure and function.
Although the availability of a wide variety of naturally occurring fluorescent proteins and spectral variants of the proteins has allowed for substantial advances, limitations to the use of fluorescent proteins remain. In particular, GFP and its spectral variants, as well as other naturally occurring fluorescent proteins such as dsRed have a propensity to self-associate under physiological conditions, thus forming dimers, tetramers, and the like. As such, it can be difficult in some cases to confirm whether a result is due, for example, to a specific interaction of two proteins under investigation, or whether a perceived interaction is an artifact caused by the oligomerization of fluorescent proteins linked to each of the two proteins under investigation.
Substantial progress has been made in designing mutants of GFP and its spectral variants that have substantially reduced oligomerizing activity. Progress also has been made in designing dsRed mutants that have a reduced propensity to form oligomers, and dsRed mutants that form only dimers have been developed. However, previous efforts to modify dsRed to prevent dimer formation have resulted in the formation of non-fluorescent proteins. Thus, a need exists for methods to reduce the propensity of red fluorescent proteins such as dsRed to self-associate. The present invention satisfies this need and provides additional advantages.